With-No-Lysine Kinase 4 (WNK4) plays an important role in regulating Na and K transport in the aldosterone-sensitive distal nephron (ASDN). Mutation of WNK4 causes familial hyperkalemic hypertension, an autosomal dominant disease characterized by salt-sensitive hypertension, hyperkalemia and renal tubule acidosis (Pseudohypoaldosteronism type II). A large body of evidence shows that WNK4 inhibits ROMK, regulates ENaC and modulates the paracellular Cl permeability in the collecting duct. The inhibitory effect of WNK4 on ROMK is attenuated by serum-glucocorticoid- induced kinase1 (SGK1)-mediated phosphorylation of WNK4 in the switch-domain. Recently, we have further demonstrated that Src-family protein tyrosine kinases (SFK), such as c-Src, phosphorylates WNK4 and diminishes SGK1-induced phosphorylation of WNK4 thereby restoring the inhibitory effect of WNK4 on ROMK channels. The interaction among WNK4, SFK and SGK1 plays an important role in stimulating K secretion during increasing K intake and in preventing K loss during volume depletion. However, the molecular mechanism by which c-Src modulates the interaction of SGK1 with WNK4 is not understood. The goal of the present proposal is to test the hypothesis that SFK-mediated phosphorylation of WNK4 activates protein phosphatase 1 (PP1) thereby abolishing the SGK1-induced stimulation of WNK4 phosphorylation and restoring the inhibitory effect of WNK4 on ROMK1. Specific Aim 1 is to test the hypothesis that c-Src binds and phosphorylates WNK4 and that the Tyr phosphorylation of WNK4 abolishes SGK1-induced phosphorylation at WNK4's switch domain thereby locking WNK4 in inhibitory mode for ROMK. Specific Aim 2 is to test the hypothesis that PP1 binds to WNK4 and is involved in mediating the effect of c-Src on WNK4-induced inhibition of ROMK. Specific Aim 3 is to test the hypothesis that PTP1D binds to WNK4, modulates PP1 activity and participates in the interaction among SGK1, WNK4 and c- Src, a mechanism which specifically regulates ROMK but has no effect on ENaC. The significance of the project is to expand the current understanding regarding the regulation of Na and K transport in the ASDN and to provide an integrated mechanism of WNK4-mediated regulation of ROMK in the ASDN.